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      Ecological Patterns of nifH Genes in Four Terrestrial Climatic Zones Explored with Targeted Metagenomics Using FrameBot, a New Informatics Tool


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          Biological nitrogen fixation is an important component of sustainable soil fertility and a key component of the nitrogen cycle. We used targeted metagenomics to study the nitrogen fixation-capable terrestrial bacterial community by targeting the gene for nitrogenase reductase ( nifH). We obtained 1.1 million nifH 454 amplicon sequences from 222 soil samples collected from 4 National Ecological Observatory Network (NEON) sites in Alaska, Hawaii, Utah, and Florida. To accurately detect and correct frameshifts caused by indel sequencing errors, we developed FrameBot, a tool for frameshift correction and nearest-neighbor classification, and compared its accuracy to that of two other rapid frameshift correction tools. We found FrameBot was, in general, more accurate as long as a reference protein sequence with 80% or greater identity to a query was available, as was the case for virtually all nifH reads for the 4 NEON sites. Frameshifts were present in 12.7% of the reads. Those nifH sequences related to the Proteobacteria phylum were most abundant, followed by those for Cyanobacteria in the Alaska and Utah sites. Predominant genera with nifH sequences similar to reads included Azospirillum, Bradyrhizobium, and Rhizobium, the latter two without obvious plant hosts at the sites. Surprisingly, 80% of the sequences had greater than 95% amino acid identity to known nifH gene sequences. These samples were grouped by site and correlated with soil environmental factors, especially drainage, light intensity, mean annual temperature, and mean annual precipitation. FrameBot was tested successfully on three ecofunctional genes but should be applicable to any.


          High-throughput phylogenetic analysis of microbial communities using rRNA-targeted sequencing is now commonplace; however, such data often allow little inference with respect to either the presence or the diversity of genes involved in most important ecological processes. To study the gene pool for these processes, it is more straightforward to assess the genes directly responsible for the ecological function (ecofunctional genes). However, analyzing these genes involves technical challenges beyond those seen for rRNA. In particular, frameshift errors cause garbled downstream protein translations. Our FrameBot tool described here both corrects frameshift errors in query reads and determines their closest matching protein sequences in a set of reference sequences. We validated this new tool with sequences from defined communities and demonstrated the tool’s utility on nifH gene fragments sequenced from soils in well-characterized and major terrestrial ecosystem types.

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          Most cited references19

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          Amino acid substitution matrices from protein blocks.

          Methods for alignment of protein sequences typically measure similarity by using a substitution matrix with scores for all possible exchanges of one amino acid with another. The most widely used matrices are based on the Dayhoff model of evolutionary rates. Using a different approach, we have derived substitution matrices from about 2000 blocks of aligned sequence segments characterizing more than 500 groups of related proteins. This led to marked improvements in alignments and in searches using queries from each of the groups.
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            The microbial pan-genome.

            A decade after the beginning of the genomic era, the question of how genomics can describe a bacterial species has not been fully addressed. Experimental data have shown that in some species new genes are discovered even after sequencing the genomes of several strains. Mathematical modeling predicts that new genes will be discovered even after sequencing hundreds of genomes per species. Therefore, a bacterial species can be described by its pan-genome, which is composed of a "core genome" containing genes present in all strains, and a "dispensable genome" containing genes present in two or more strains and genes unique to single strains. Given that the number of unique genes is vast, the pan-genome of a bacterial species might be orders of magnitude larger than any single genome.
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              Nitrogenase gene diversity and microbial community structure: a cross-system comparison.

              Biological nitrogen fixation is an important source of fixed nitrogen for the biosphere. Microorganisms catalyse biological nitrogen fixation with the enzyme nitrogenase, which has been highly conserved through evolution. Cloning and sequencing of one of the nitrogenase structural genes, nifH, has provided a large, rapidly expanding database of sequences from diverse terrestrial and aquatic environments. Comparison of nifH phylogenies to ribosomal RNA phylogenies from cultivated microorganisms shows little conclusive evidence of lateral gene transfer. Sequence diversity far outstrips representation by cultivated representatives. The phylogeny of nitrogenase includes branches that represent phylotypic groupings based on ribosomal RNA phylogeny, but also includes paralogous clades including the alternative, non-molybdenum, non-vanadium containing nitrogenases. Only a few alternative or archaeal nitrogenase sequences have as yet been obtained from the environment. Extensive analysis of the distribution of nifH phylotypes among habitats indicates that there are characteristic patterns of nitrogen fixing microorganisms in termite guts, sediment and soil environments, estuaries and salt marshes, and oligotrophic oceans. The distribution of nitrogen-fixing microorganisms, although not entirely dictated by the nitrogen availability in the environment, is non-random and can be predicted on the basis of habitat characteristics. The ability to assay for gene expression and investigate genome arrangements provides the promise of new tools for interrogating natural populations of diazotrophs. The broad analysis of nitrogenase genes provides a basis for developing molecular assays and bioinformatics approaches for the study of nitrogen fixation in the environment.

                Author and article information

                American Society of Microbiology (1752 N St., N.W., Washington, DC )
                17 September 2013
                Sep-Oct 2013
                : 4
                : 5
                : e00592-13
                Center for Microbial Ecology, Michigan State University, East Lansing, Michigan, USA [ a ]
                School of Civil and Environmental Engineering, Yonsei University, Seoul, Republic of Korea [ b ]
                Computer Science and Engineering Department, Michigan State University, East Lansing, Michigan, USA [ c ]
                Author notes
                Address correspondence to James M. Tiedje, tiedjej@ 123456msu.edu .

                Editor Jizhong Zhou, University of Oklahoma

                Copyright © 2013 Wang et al.

                This is an open-access article distributed under the terms of the Creative Commons Attribution-Noncommercial-ShareAlike 3.0 Unported license, which permits unrestricted noncommercial use, distribution, and reproduction in any medium, provided the original author and source are credited.

                : 5 August 2013
                : 16 August 2013
                Page count
                Pages: 9
                Research Article
                Custom metadata
                September/October 2013

                Life sciences
                Life sciences


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